Structure of the O-specific polysaccharide of Proteus vulgaris O45 containing 3-acetamido-3,6-dideoxy-d-galactose

Eur. J. Biochem. 240, 245-251 (1996) 0 FEBS 1996

The structure of the 0-specific polysaccharide of Proteus penneri 52 Zygmunt SIDORCZYK', Krystyna ZYCH', Anna SWIERZK02, Eugeny V. WNOGRADOV' and Yuriy A. KNIREL3 I

Institute of Microbiology and Immunology, University of t o d i , Poland Center of Microbiology and Virology, Polish Academy of Sciences, G d i , Poland N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia

(Received 23 ApriU28 June 1996)

-

EJB 96 0603/5

An acidic 0-specific polysaccharide isolated from Proteus penneri 52 contains D-galacturonic acid, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-~-galactose in the ratio 1 :2 :2. On the basis of sugar analysis and NMR spectroscopy, which included one-dimensional TOCSY, two-dimensional COSY, heteronuclear ''C, 'H-COSY, and rotating-frame NOE spectroscopy, the following structure of the pentasaccharide repeating unit of the 0-specific polysaccharide was established: P-D-G~c~NAc

1

1 3 -4)-P-~-GalpA-( 1+3)-P-~-GlcpNAc-(1+4)-p-~-GalpNAc-(1+3)-a-D-GalpNAc-( 1Cross-reactivity of the anti-P-penneri 52 0-serum with other strains of l? penneri isolated in Germany, Poland, USA, and Canada is discussed and a new Proteus serogroup is proposed.

Keywords: Proteis penneri; 0-antigen serology ; epitope; polysaccharide structure; lipopolysaccharide.

Recently, 0-antigens of several strains of a newly described species Proteus penneri have been structurally elucidated and found to be acidic polysaccharides containing D-glucuronic acid [l],D-galacturonic acid (D-GalA) [2],amides of D-GalA with Lalanine [3] or L-threonine [4], and isomers of N-acetylmuramic acid : 2-acetarnido-3-O-[(S)-l -carboxyethyl]-2-deoxy-~-glucose (N-acetylisomurarnic acid) [ 5 ] and 2-acetainido-4-O-[(S)-l -carboxyethyl]-2-deoxy-~-glucose [6]. We now describe the structure of another acidic 0-specific polysaccharide containing D-GalA, which was isolated from Iipopolysaccharide (LPS) of l? penneri 52.

EXPERIMENTAL PROCEDURES Miscellaneous methods. Optical rotations were measured with a Jasco DIP 360 polarimeter in water at 25°C. 'H-NMR and 'T-NMR spectra of the polysaccharide were recorded with a Bruker AM-300 instrument in D,O at 70°C. Acetone was used as internal standard (6, 2.225, 8, 31.45). One-dimensional TOCSY, two-dimensional COSY, heteronuclear ''C, 'H-COSY, and rotating-frame NOE spectroscopy (ROESY) were performed as described [I, 71. Gel-permeation chromatography was carried out on a column (40 cmX2.5 cm) of Sephadex G-50 in pyridine acetate, pH 4.5, and monitored by the phenol/sulfuric acid reaction. Ascending paper chromatography was carried out on FN-I1 paper Correspondence to 2. Sidorczyk, Institute of Microbiology and Immunology, University of t 6 d i . Banacha 12/16, PL-90-237 t 6 d i , Poland Abbreviations. GdlA, galacturonic acid; LPS, lipopolysaccharide; ROESY, rotating-frame NOE spectroscopy.

in a system of 5 : 5 :1: 3 (by vol.) pyridine/ethyl acetate/acetic acid/water with detection of amino sugars by ninhydrin.

Bacterial strains, growth of bacteria, isolation of LPS, and isolation of 0-specific polysaccharide. l? penneri 52 was one of the nine strains (P: penneri 50-58) kindly provided by Dr E. J. Wolter (Institute of Epidemiology, Wernigerode, Germany). These strains, together with the strains l? penneri 61-75 isolated in Poland, compose the European collection. For comparative serological investigation, 45 strains of l? penneri, isolated in the USA and Canada (a gift from Prof. D. J. Brenner, Center for Disease Control, Atlanta, USA), and strains of Proteus rnirubilis 0 2 7 and 0 2 8 from the Czechoslovak National Collection of Type Cultures were used. Dry bacterial cells were obtained from aerated liquid cultures as described [8]. Isolation of LPS by the phenol/water procedure 191 and isolation of 0-specific polysaccharide by mild degradation of LPS with diluted acetic acid followed by gel-permeation chromatography on Sephadex G-50 [lo] were performed as described previously. The polysaccharide was 0-deacetylated with 12% aqueous ammonia (50"C, 2 h). Sugar analysis. The polysaccharide was hydrolysed with 2 M trifluoroacetic acid (121 "C, 2 h), and the hydrolysate was analysed using a Biotronik LC 2000 sugar and amino acid analyser as described previously [I]. Solvolysis of the polysaccharide with anhydrous hydrogen fluoride was performed at 25 "C for 1 h; GalA was isolated by anion-exchange Chromatography on a column (10 cmXl cm) of Dowex 1 x 8 using a linear gradient of aqueous acetic acid (0-10%) and chromatography was monitored by the orcinol/sulfuric acid reaction. N-acetylated amino sugars were hydrolysed with 2 M hydrochloric acid (100"C, 2 h) and separated by preparative paper chromatogra-

246

Sidorczyk et al. ( E M J. Biochem. 240)

PPM

Chemical shift (ppm)

Fig. 1. 90-MHz "C-NMR spectrum of P. penneri 52 0-specific polysaccharide.

Table 1. "C-NMR data for the 0-specific polysaccharide. Chemical shifts for the N-acetyl groups are 23.7-24.0 ppm (Me). Assignments of the signals for CO of the N-acetyl groups (tentatively at 174.5-175.5 ppm) and C6 of GalA (unit D) could be interchanged. Chemical shift for

Poly saccharide

~

c1

c2

C3

c4

c5

-3)-/h-GlcpNAc-(l (unit A) +3)-a-~-GalpNAc-(l- (unit B) jl-~-GlcpNAc-(l- (unit C)

101.9 100.4 103.8

55.3 49.7 57.1

86.2 77.6 74.9

70.7 70.0 71.7

77.2 12 2 77.0

n\ U )

i1nV-f.0 n Q

79 n I L.,,

7I d.7 2n

an 2 "".J

I, J